Our long-term goal is to understand how the fate of proteins is regulated by the ubiquitin (Ub) system. Ub, an abundant 76-residue protein, is highly conserved among eukaryotes. Ubiquitylation - the covalent conjugation of Ub to lysine residues on other intracellular proteins - regulates a myriad of cellular processes, including cell cycle progression, DNA repair, transcription, stress responses and signal transduction. Ub is best known as a signal to target proteins for destruction by a multisubunit, ATP dependent protease termed the proteasome. How the substrates are delivered to the proteasome is one of the most challenging issues in the field. It is proposed that adaptor molecules, which selectively recognize ubiquitylated substrates, perform this vital function in deciding the final destination of substrates. We will focus on S. cerevisiae Rad23, a candidate adaptor molecule involved in delivering ubiquitylated substrates to the proteasome. Rad23 has two functional domains: a ubiquitin-like element (UBL), and a ubiquitin-associated motif (UBA). The UBL motif was shown to directly bind the proteasome subunit Rpn1. Several groups including ours found that the UBA domain preferentially binds ubiquitylated substrates. And yeast cells lacking Rad23 are deficient in proteolysis. Importantly, Rad23 promotes the formation of the proteasome-Ub conjugates complex in vivo and in vitro. More recently, we found that Rad23 and Dsk2 interact with Ufd2, an E4 enzyme important for Ub-chain assembly. Based on biochemical properties and genetic evidence, we propose that Rad23-like adaptor proteins recognize multi-ubiquitylated substrates and deliver them to the proteasome through various binding partners. The yeast Ufd2-Rad23 complex regulates the degradation of UFD substrates, Hmg-CoA reductase, and the transcription factor Spt23, and prion protein. We propose the following aims in an effort to decipher the biological role of the adaptor molecules in substrate proteolysis. Aim 1 is to understand the mechanism underlying the substrate selectivity of Rad23. Aim 2 is to determine the regulation of the Rad23-Ufd2 complex. Aim 3 is to define the function of Rad23-mediated proteolysis in prion biogenesis. These studies should reveal novel insights into the mechanisms and functions of the Ub system, and provide defined molecular targets for future intervention in human diseases.